Protocol for the Long-Term Cryopreservation of Carotenoid-Producing Rhodotorula spp. Strains.

The genus Rhodotorula comprises oleaginous yeasts widely distributed across diverse environments and recognized for their ability to synthesize carotenoids, lipids, and other high-value metabolites with biotechnological relevance. The preservation of these microorganisms is critical to maintain their genetic stability, metabolic potential, and phenotypic traits over time. Among available methods, cryopreservation using glycerol is one of the most effective strategies for long-term storage, as it protects cellular structures during freezing and minimizes metabolic activity at ultra-low temperatures. In this protocol, we describe a standardized and reproducible method for the long-term cryopreservation of the carotenoid-producing strain, Rhodotorula glutinis. The procedure includes cultivation under controlled conditions, preparation of cell suspensions in glycerol, stabilization at 4°C, controlled-rate freezing, and storage at -80°C. Post-reactivation is achieved by direct streaking from frozen stocks, followed by incubation and evaluation of colony morphology and purity. This protocol ensures high postthaw viability and preservation of key phenotypic characteristics, providing a reliable approach for maintaining Rhodotorula strains from diverse origins. Its application supports the establishment of microbial collections and facilitates future research and biotechnological exploitation of these yeasts.